Collective migration of epithelial cells is essential for embryonic development, wound healing and the spread of some cancers. For an epithelium to migrate, the cytoskeletal machinery that powers each cell?s motility must become globally aligned across the tissue. It is likely that diverse cell-cell and cell-matrix signals are integrated to achieve this goal. However, few of these signals have been identified and even fewer are understood at a mechanistic level. The goal of this research is to define the signals that orient the migration machinery for epithelial motility, and to use this knowledge to generate new ideas for wound healing and cancer therapies. To this end, we are using genetic, cell biological, and high resolution live imaging approaches to study a dramatic collective migration that occurs in the Drosophila follicular epithelium. The cytoskeletal machinery that powers this tissue?s movement shows a particularly high degree of global alignment, a pattern that is best seen through the alignment of contractile actin stress fibers across the tissue. Because stress fibers generate traction forces for individual cell motility though their interaction with integrin-based adhesions, their global alignment is essential for efficient collective movement. Aligned stress fibers are also seen in mammals, with notable examples being the collective migrations of endothelial cells and the epithelial cells of the cornea and lens. However, little is known about how this tissue-level actin network is generated. The Specific Aims in this proposal will explore how three distinct signals impact different aspects of the stress fiber alignment program: initiation, reinforcement/stabilization, and local disassembly. Aim 1 will test the hypothesis that cell protrusive activity, as induced by signaling from the Fat2 cadherin, can initiate global stress fiber alignment as migration begins. Aim 2 will investigate whether a novel polarized feature within the basement membrane (BM) ECM that we have termed BM ridges reinforces and/or stabilizes the global stress fiber pattern. Finally, Aim 3 explores whether a newly identified semaphorin signal helps to control the local disassembly of stress fibers that is required for forward movement to occur. Our discovery of the semaphorin?s role in epithelial motility, as well as the key entry points for Aims 1 and 2, all came from a small-scale genetic screen. Thus, to continue to gain novel insights into the signaling mechanisms that promote epithelial motility, work proposed in Aim 3 will also extend our successful screening strategy to other regions of the genome. The signals we propose to study have been implicated in epithelial migrations in mammals, but how they promote collective motility is unknown. Thus, by defining the cell-cell and cell-matrix signals that generate and maintain the tissue-level stress fiber pattern in the follicular epithelium, we will provide a general paradigm for understanding how an epithelium becomes polarized for directed migration in a variety of contexts.